U.S. patent number 5,635,156 [Application Number 08/337,785] was granted by the patent office on 1997-06-03 for non-lethal methods for conditioning a recipient for bone marrow transplantation.
This patent grant is currently assigned to University of Pittsburgh. Invention is credited to Suzanne T. Ildstad.
United States Patent |
5,635,156 |
Ildstad |
June 3, 1997 |
Non-lethal methods for conditioning a recipient for bone marrow
transplantation
Abstract
The present invention relates to non-lethal methods of
conditioning a recipient for bone marrow transplantation. In
particular, it relates to the use of nonlethal doses of total body
irradiation, total lymphoid irradiation cell type-specific
antibodies, especially antibodies directed to bone marrow stromal
cell markers, cytotoxic drugs, or a combination thereof. The
methods of the invention have a wide range of applications,
including, but not limited to, the conditioning of an individual
for hematopoietic reconstitution by bone marrow transplantation for
the treatment of hematologic malignancies, hematologic disorders,
autoimmunity, infectious diseases such as acquired immunodeficiency
syndrome, and the engraftment of bone marrow cells to induce
tolerance for solid organ, tissue and cellular transplantation.
Inventors: |
Ildstad; Suzanne T.
(Pittsburgh, PA) |
Assignee: |
University of Pittsburgh
(Pittsburgh, PA)
|
Family
ID: |
26818205 |
Appl.
No.: |
08/337,785 |
Filed: |
November 14, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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120256 |
Sep 13, 1993 |
5514364 |
|
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Current U.S.
Class: |
424/1.49;
424/130.1; 424/141.1; 424/152.1; 424/153.1; 424/154.1; 424/178.1;
424/181.1; 424/183.1; 600/1; 604/20 |
Current CPC
Class: |
A61K
41/00 (20130101); A61K 47/6803 (20170801); A61K
47/68 (20170801) |
Current International
Class: |
A61K
41/00 (20060101); A61K 47/48 (20060101); A61K
043/00 (); A61K 031/00 (); A61K 005/00 () |
Field of
Search: |
;424/1.49,1.53,130.1,141.1,178.1,183.1,152.1,153.1,154.1,181.1
;600/1,9,13,14 ;604/20 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Down et al., "Synogeneic and Allogeneic Bone Marrow Engraftment
After Total Body Irradiation," Blood, 77(3), Jan. 1, 1991, pp.
661-669. .
Pierce et al., "Effects of Thy-1.sup.+ Cell Depletion . . . ",
Transplantation, 48(2), Aug. 1989, pp. 289-296. .
Gassmann et al., "Immune Reactivity After High-Dose Irradiation,"
Transplantation, 41(3), Mar. 1986, pp. 380-384. .
Pierce et al., "The Role of Donor Lymphoid Cells . . . ",
Transplantation, 40(6), Dec. 1985, pp. 702-707. .
Nakamura et al., "Graft Rejection by Cytolytic T Cells . . . ,"
Transplantation, 49(2), Feb. 1990, pp. 453-458. .
Antin et al., "Selective Depletion of Bone Marrow T Lymphocytes . .
. ," Blood, 78(8), 1991, pp. 2139-2149, abstracted in Biosis
92:6551..
|
Primary Examiner: Kight; John
Assistant Examiner: Chapman; Lara E.
Attorney, Agent or Firm: Pennie & Edmonds
Parent Case Text
The present application is a continuation-in-part of application
Ser. No. 08/120,256 filed Sep. 13, 1993 now U.S. Pat. No.
5,514,364, which is incorporated by reference herein in its
entirety.
Claims
What is claimed is:
1. A method for conditioning a recipient for bone marrow
transplantation comprising subjecting the recipient to treatment
with a non-lethal dose of total body irradiation, an alkylating
agent and an antibody or an active fragment thereof, followed by
transplantation with a donor cell preparation containing
hematopoietic stem cells which are not compatible with the
recipient at the major histocompatibility complex, to achieve
stable engraftment of donor hematopoietic stem cells.
2. The method of claim 1 in which the dose is between 1 Gy and 7
Gy.
3. The method of claim 1 in which the alkylating agent is
cyclophosphamide.
4. The method of claim 1 in which the antibody is anti-lymphocyte
globulin.
5. The method of claim 1 in which the antibody is of monoclonal
origin.
6. The method of claim 5 in which the antibody is conjugated to an
anti-proliferative agent.
7. The method of claim 6 in which the anti-proliferative agent is a
chemotherapeutic drug.
8. The method of claim 6 in which the anti-proliferative agent is a
radioactive isotope.
9. The method of claim 6 in which the anti-proliferative agent is a
toxin.
10. The method of claim 1 in which the antibody is reactive with a
bone marrow stromal cell.
Description
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. NON-LETHAL CONDITIONING REGIMENS FOR DONOR CELL
ENGRAFTMENT
5.2. ANTIBODY FOR USE IN CONDITIONING
5.3. USES OF ANTIBODIES TO STROMAL CELLS
6. EXAMPLE: ALLOGENEIC BONE MARROW CELLS ENGRAFT IN RECIPIENTS
CONDITIONED BY NON-LETHAL METHODS
6.1. MATERIALS AND METHODS
6.1.1. ANIMALS
6.1.2. FLOW CYTOMETRY
6.1.3. PLATELET ISOLATION
6.1.4. GLUCOSE PHOSPHATE ISOMERASE-1 (GPI-1) ASSAY
6.1.5. SKIN GRAFTING
6.1.6. MIXED LYMPHOCYTE REACTIONS (MLR)
6.1.7. CELL-MEDIATED LYMPHOLYSIS (CML)
6.2. RESULTS
6.2.1. ALLOGENEIC ENGRAFTMENT WITH NONLETHAL TOTAL BODY IRRADIATION
ALONE: DOSE-TITRATION OF RADIATION-BASED CONDITIONING
6.2.2. ENGRAFTMENT OF ALLOGENEIC BONE MARROW IS ENHANCED BY
ANTI-LYMPHOCYTE GLOBULIN
6.2.3. INFLUENCE OF CELL DOSE IN THEALLOGENEIC INOCULUM ON
ENGRAFTMENT WITH ALG AND TBI CONDITIONING
6.2.4. ALLOGENEIC ENGRAFTMENT IS ENHANCED BY THE ADDITION OF
CYCLOPHOSPHAMIDE TO THE ESTABLISHED RADIATION-BASED
CONDITIONING
6.2.5. INFLUENCE OF TIMING OF TBI ON ALLOENGRAFTMENT IN RECIPIENTS
CONDITIONED WITH ANTI-LYMPHOCYTE GLOBULIN OR CYCLOPHOSPHAMIDE
6.2.6. CHARACTERIZATION OF A NONLETHAL RADIATION-BASED APPROACH FOR
CYTOREDUCTION
6.2.7. NONLETHAL MIXED CHIMERAS: EVIDENCE FOR MULTILINEAGE MIXED
CHIMERISM
6.2.8. EVIDENCE THAT ERYTHROCYTES AND PLATELETS IN ALLOGENEIC
CHIMERAS ARE OF BOTH SYNGENEIC AND ALLOGENEIC ORIGIN
6.2.9. EVIDENCE FOR SPECIFIC TOLERANCE IN VIVO TO DONOR-TYPE SKIN
GRAFTS
6.2.10. FUNCTIONAL DONOR-SPECIFIC TOLERANCE IN VITRO
6.2.11. NONLETHAL PREPARATIVE REGIMENS RESULT IN STABLE ALLOGENEIC
CHIMERISM AND EXCELLENT LONG-TERM RECIPIENT SURVIVAL AND NO
EVIDENCE FOR GVHD
6.2.12. ALLOGENEIC ENGRAFTMENT AFTER CONDITIONING WITH NONLETHAL
TOTAL BODY IRRADIATION, ANTI-LYMPHOCYTE GLOBULIN AND
CYCLOPHOSPHAMIDE
7. EXAMPLE: XENOGENEIC BONE MARROW CELLS ENGRAFT IN RECIPIENTS
CONDITIONED BY NON-LETHAL METHODS
7.1. RESULTS
1. INTRODUCTION
The present invention relates to non-lethal methods of conditioning
a recipient for bone marrow transplantation. In particular, it
relates to the use of nonlethal doses of total body irradiation,
total lymphoid irradiation, cell type-specific antibodies,
especially antibodies directed to bone marrow stromal cell markers,
cytotoxic drugs, or a combination thereof. The methods of the
invention have a wide range of applications, including, but not
limited to, the conditioning of an individual for hematopoietic
reconstitution by bone marrow transplantation for the treatment of
hematologic malignancies, hematologic disorders, autoimmunity,
infectious diseases such as acquired immunodeficiency syndrome, and
the engraftment of bone marrow cells to induce tolerance for solid
organ, tissue and cellular transplantation.
2. BACKGROUND OF THE INVENTION
A major goal in solid organ transplantation is the permanent
engraftment of the donor organ without a graft rejection immune
response generated by the recipient, while preserving the
immunocompetence of the recipient against other foreign antigens.
Typically, in order to prevent host rejection responses,
nonspecific immunosuppressive agents such as cyclosporine,
methotrexate, steroids and FK506 are used. These agents must be
administered on a daily basis and if stopped, graft rejection
usually results. However, a major problem in using nonspecific
immunosuppressive agents is that they function by suppressing all
aspects of the immune response, thereby greatly increasing a
recipient's susceptibility to infections and other diseases,
including cancer.
Furthermore, despite the use of immunosuppressive agents, graft
rejection still remains a major source of morbidity and mortality
in human organ transplantation. Most human transplants fail within
10 years without permanent graft acceptance. Only 50% of heart
transplants survive 5 years and 20% of kidney transplants survive
10 years. (See Opelz et al., 1981, Lancet 1: 1223; Gjertson, 1992,
UCLA Tissue Typing Laboratory, p. 225; Powles, 1980, Lancet, p.
327; Ramsay, 1982, New Engl. J. Med., p. 392). It would therefore
be a major advance if tolerance to the donor cells can be induced
in the recipient.
The only known clinical condition in which complete systemic
donor-specific transplantation tolerance occurs is when chimerism
is created through bone marrow transplantation. (See Qin et al.,
1989, J Exp Med. 169: 779; Sykes et al., 1988, Immunol. Today 9:
23; Sharabi et al., 1989, J. Exp. Med. 169: 493). This has been
achieved in neonatal and adult animal models as well as in humans
by total lymphoid irradiation of a recipient followed by bone
marrow transplantation with donor cells. The success rate of
allogeneic bone marrow transplantation is, in large part, dependent
on the ability to closely match the major histocompatibility
complex (MHC) of the donor cells with that of the recipient cells
to minimize the antigenic differences between the donor and the
recipient, thereby reducing the frequency of host-versus-graft
responses and graft-versus-host disease (GVHD). In fact, MHC
matching is essential, only one or two antigen mismatch is
acceptable because GVHD is very severe in cases of greater
disparities. In addition, it also requires the appropriate
conditioning of the recipient by lethal doses of total body
irradiation (TBI).
The MHC is a gene complex that encodes a large array of
individually unique glycoproteins expressed on the surface of both
donor and host cells that are the major targets of transplantation
rejection immune responses. In the human, the MHC is referred to as
HLA. When HLA identity is achieved by matching a patient with a
family member such as a sibling, the probability of a successful
outcome is relatively high, although GVHD is still not completely
eliminated. However, when allogeneic bone marrow transplantation is
performed between two MHC-mismatched individuals of the same
species, common complications involve failure of engraftment, poor
immunocompetence and a high incidence of GVHD. Unfortunately, only
about 20% of all potential candidates for bone marrow
transplantation have a suitable family member match.
The field of bone marrow transplantation was developed originally
to treat bone marrow-derived cancers. It is believed by those
skilled in the art even today that lethal conditioning of a human
recipient is required to achieve successful engraftment of donor
bone marrow cells in the recipient. In fact, prior to the present
invention, current conventional bone marrow transplantation has
exclusively relied upon lethal conditioning approaches to achieve
donor bone marrow engraftment. The requirement for lethal
irradiation of the host which renders it totally immunocompetent
poses a significant limitation to the potential clinical
application of bone marrow transplantation to a variety of disease
conditions, including solid organ or cellular transplantation,
sickle cell anemia, thalassemia and aplastic anemia.
The risk inherent in tolerance-inducing conditioning approaches
must be low when less toxic means of treating rejection are
available or in cases of morbid, but relatively benign conditions.
In addition to solid organ transplantation, hematologic disorders,
including aplastic anemia, severe combined immunodeficiency (SCID)
states, thalassemia, diabetes and other autoimmune disease states,
sickle cell anemia, and some enzyme deficiency states, may all
significantly benefit from a nonlethal preparative regimen which
would allow partial engraftment of allogeneic or even xenogeneic
bone marrow to create a mixed host/donor chimeric state with
preservation of immunocompetence and resistance to GVHD. For
example, it is known that only approximately 40% of normal
erythrocytes are required to prevent an acute sickle cell crisis
(Jandl et al., 1961, Blood 18(2): 133; Cohen et al., 1984, Blood
76(7): 1657), making sickle cell disease a prime candidate for an
approach to achieve mixed multilineage chimerism. Although the
morbidity and mortality associated with the conventional full
cytoreduction currently utilized for allogeneic bone marrow
transplantation cannot be justified for relatively benign
disorders, the induction of multilineage chimerism by a less
aggressive regimen certainly remains a viable option. Moreover, the
use of bone marrow from an HIV-resistant species offers a potential
therapeutic strategy for the treatment of acquired immunodeficiency
syndrome (AIDS) if bone marrow from a closely related species will
also engraft under similar nonlethal conditions, thereby producing
new hematopoietic cells such as T cells which are resistant to
infection by the AIDS virus.
A number of sublethal conditioning approaches in an attempt to
achieve engraftment of allogeneic bone marrow stem cells with less
aggressive cytoreduction have been reported in rodent models
(Mayumi and Good, 1989, J Exp Med 169: 213; Slavin et al., 1978, J
Exp Med 147(3): 700; McCarthy et al., 1985, Transplantation 40(1):
12; Sharabi et al., 1990, J Exp Med 172(1): 195; Monaco et al.,
1966, Ann NY Acad Sci 129: 190). However, reliable and stable donor
cell engraftment as evidence of multilineage chimerism was not
demonstrated, and long-term tolerance has remained a question in
many of these models (Sharabi and Sachs, 1989, J. Exp. Med. 169:
493; Cobbold et al., 1992, Immunol. Rev. 129: 165; Qin et al.,
1990, Eur. J. Immunol. 20: 2737). Moreover, reproducible
engraftment has not been achieved, especially when multimajor and
multiminor antigenic disparities existed.
Permanent tolerance to donor antigens has been documented in H-2
(MHC) identical or congenic strains with minimal therapy and/or
transplantation of donor skin drafts or splenocytes alone (Qin et
al., 1990, Eur J Immunol 20: 2737). However, similar attempts to
achieve engraftment and tolerance in MHC-mismatched combinations
have not enjoyed the same success. In most models, only transient
donor-specific tolerance has been achieved (Mayumi et al., 1987,
Transplantation 44(2): 286; Mayumi et al., 1986, Transplantation
42(4): 417; Cobbold et al., 1990, Eur J Immunol 20: 2747; Cobbold
et al., 1990, Seminars in Immunology 2: 377).
Early work by Wood and Monaco attempted to induce tolerance using
bone marrow plus anti-lymphocyte serum (ALS) in partial MHC-matched
donor-recipient combinations (Wood et al., 1971, Trans Proc 3(1):
676; Wood and Monaco, 1977, Transplantation (Baltimore) 23: 78).
Even in this semi-allogeneic system, F.sub.1 splenocytes were
required to facilitate the induction of tolerance, and thymectomy
was required for stable long-term tolerance. The additional
requirement for splenocytes and thymectomy made potential clinical
applicability of such an approach unlikely. However, these studies
identified two key factors required for the induction of tolerance:
an antigenic source of tolerogen, which is not only involved in
tolerance induction, but must also be present at least periodically
for permanent antigen-specific tolerance, and a method to tolerize,
or prevent activation of new T cells from the thymus, i.e.
thymectomy, or intrathymic clonal deletion.
Attempts to induce tolerance to allogeneic bone marrow donor cells
using combinations of depleting and non-depleting anti-CD4 and CD8
monoclonal antibodies (mAb) resulted in only transient tolerance to
MHC-compatible combinations (Cobbold et al., 1992, Immunol Rev 129:
165; Qin et al., 1990, Eur J Immunol 20: 2737). 6Gy of TBI was
required to obtain stable engraftment and tolerance when
MHC-disparate bone marrow was utilized (Cobbold et al., 1986,
Transplantation 42: 239). Sharabi and Sachs attributed the failure
of anti-CD4/CD8 mAb therapy alone to the inability of mAb to
deplete T cells from the thymus, since persistent cells coated with
mAb could be identified in this location (Sharabi and Sachs, 1989,
J Exp Med 169: 493). However, subsequent attempts to induce
tolerance by the addition of 7Gy of selective thymic irradiation
prior to donor bone marrow transplantation also failed. Engraftment
was only achieved with the addition of 3Gy of recipient TBI.
Therefore, there remains a need for non-lethal methods of
conditioning a recipient for allogeneic bone marrow transplantation
that would result in stable mixed multilineage allogeneic chimerism
and long-term donor-specific tolerance.
3. SUMMARY OF THE INVENTION
The present invention relates to non-lethal methods of conditioning
a recipient for bone marrow transplantation. These methods include
the use of non-lethal doses of irradiation, cell type-specific
antibodies and active fragments thereof, cytotoxic drugs or a
combination thereof.
The invention is based, in part, on the Applicant's discovery that
treatment of normal mice with non-lethal doses of TBI permits the
engraftment of allogeneic bone marrow cells in virtually all
recipients. In addition, the dosage of TBI can be further reduced
when used in combination with anti-lymphocyte globulin (ALG) or an
alkylating agent such as cyclophosphamide (CyP). The dosage of TBI
can be reduced even more if it is used with both ALG and CyP,
agents with different mechanisms of action and non-overlapping
toxicities. The reconstituted animals exhibit stable mixed
multilineage chimerism in their peripheral blood containing both
donor and recipient cells of all lymphohematopoietic lineages,
including T cells, B cells, natural killer (NK) cells, macrophages,
erythrocytes and platelets. Furthermore, the mixed allogeneic
chimeras display donor-specific tolerance to donor-type skin
grafts, while they readily reject third-party skin grafts.
Donor-specific tolerance is confirmed also by in vitro assays in
which lymphocytes obtained from the chimeras are shown to have
diminished proliferative and cytotoxic activities against
allogeneic donor cells, but retain normal immune reactivity against
third-party cells. All allogeneic chimeras conditioned by
non-lethal means survive long-term, maintain stable chimerism and
do not manifest symptoms of GVHD.
The working examples further demonstrate that total lymphoid
irradiation (TLI), a less aggressive and cytoablative regimen than
TBI, may also be used at non-lethal doses to condition non-human
primates prior to allogeneic or xenogeneic bone marrow
transplantation. TLI may be used most effectively with agents such
as CyP, and/or ALG, upon optimizing engraftment with a strategy to
minimize toxicity to the recipient.
The hematopoietic microenvironment plays a major role in the
engraftment of hematopoietic stem cells. In addition to being a
source of growth factors and cellular interactions for the survival
and renewal of stem cells, it may also provide physical space for
these cells to reside. A number of cell types collectively referred
to as stromal cells are found in the vicinity of the hematopoietic
stem cells in the bone marrow microenvironment. These cells include
both bone marrow-derived CD45.sup.+ cells and non-bone
marrow-derived CD45.sup.- cells, such as adventitial cells,
reticular cells, endothelial cells and adipocytes.
Recently, the Applicant has identified another bone marrow-derived
cell type known as hematopoietic facilitatory cells, which when
co-administered with donor bone marrow cells enhance the ability of
the donor cells to stably engraft in allogeneic and xenogeneic
recipients. The facilitatory cells and the stromal cells occupy a
substantial amount of space in a recipient's bone marrow
microenvironment, which may present a barrier to donor cell
engraftment. Hematopoietic stem cells bind to facilitatory cells in
vitro and in vivo. Thus, the facilitatory cells may provide
physical space or niche on which the stem cells survive and are
nurtured. It is therefore desirable to develop conditioning
regimens to specifically target and eliminate these and other
stromal cell populations in order to provide the space necessary
for the hematopoietic stem cells and the associated facilitatory
cells in a donor cell preparation to engraft without the use of
lethal irradiation.
A wide variety of uses are encompassed by the invention described
herein, including, but not limited to, the conditioning of
recipients by non-lethal methods for bone marrow transplantation in
the treatment of diseases such as hematologic malignancies,
infectious diseases such as AIDS, autoimmunity, enzyme deficiency
states, anemias, thalassemias, sickle cell disease, and solid organ
and cellular transplantation.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Percentage of animals which engrafted with allogeneic or
xenogeneic bone marrow as a function of TBI dose. Lymphoid
chimerism was assessed by flow cytometry 2 months post
reconstitution. Donor chimerism as low as 0.5% can be detected
using this method. Data points represent results for 8 to 20
recipients pooled from 2 to 5 experiments.
FIG. 2 Percent of animals with allogeneic engraftment in mice
treated with one of three conditioning approaches prior to
allogeneic bone marrow transplantation--ALG alone given three days
prior to transplantation (n=4); 5Gy TBI alone given on the day of
transplantation (n=6); or a combination of ALG and 5Gy TBI each as
administered previously (n=16). Typing of PBL obtained from treated
animals 2 months post reconstitution (BALB/c.fwdarw.B10) was
performed using anti Class I H-2.sup.b -FITC and H-2.sup.d -FITC
mAb. Analysis was performed in the lymphoid gate and all values
were normalized to 100%.
FIG. 3 Percent of animals with allogeneic chimerism in mice treated
with one of three conditioning approaches--CyP alone given 2 days
prior to bone marrow transplantation (n =5); 5Gy TBI alone on the
day of transplantation (n=14); or 5Gy TBI given at the time of
marrow transplantation followed 2 days later by CyP (n=8). PBL
typing was performed by flow cytometry 2 months post reconstitution
(B10.BR.fwdarw.B10 and BALB/c.fwdarw.B10).
FIG. 4 Percent of mice which engrafted after conditioning with 5Gy
TBI given one week prior to BALB/c allogeneic bone marrow
transplantation. TBI was administered alone (n=4), followed by ALG
given three days prior to bone marrow transplantation (n=4), or
followed by CyP given over a four day course prior to
transplantation (n=4). Percent of animals which engrafted is
represented as a function of the recipient conditioning regimen.
PBL typing by flow cytometry was performed to assess donor
chimerism in treated animals 2 months after reconstitution. Results
are from 1 of 4 representative experiments.
FIG. 5 Life-table survival of untransplanted control mice treated
with various nonlethal conditioning regimens. Survival following
treatment with 7Gy; 6Gy; 5Gy; 5Gy plus 7 .mu.g/kg ALG i.v.; or 5Gy
plus 200 mg/kg CyP i.p. as compared to conventional 9.5Gy lethal
irradiation.
FIG. 6A-6J Two-color flow cytometric analysis for the proportion of
allogeneic donor-derived lymphoid (T and B cell), NK, and myeloid
(macrophage and granulocyte) lineages in a representative mixed
allogeneic chimera prepared using a nonlethal conditioning regimen
(BALB/c.fwdarw.B10). Splenic lymphoid tissue was analyzed 10-12
weeks following reconstitution.
Recipient (H-2.sup.b) and donor-derived (H-2.sup.d) cells of
lymphoid and NK lineages were analyzed in the lymphoid gate using
anti-H-2.sup.b and H-2.sup.d mAb directly conjugated to FITC or
biotinylated and detected with a second streptavidin antibody
conjugated to PE (SA-PE). The various subsets were analyzed using
anti-T lymphocyte mAb (.alpha..beta.TCR-PE, CD4-FITC, CD8-PE),
shown in FIG. 6A-6F, and anti-B lymphocyte (B220-FITC), and
anti-natural killer cell (NK1.1-PE) uAb displayed in
FIG. 6G-6J. FITC and PE conjugated Leu4 were used as irrelevant
controls for background staining for all flow cytometric analysis.
The percentage of donor and recipient-derived cells within each
lineage is expressed in the upper right hand corner of each
respective plot. Results are normalized to 100%.
FIG. 6K-6N Two-color flow cytometric analysis for the proportion of
allogeneic donor-derived lymphoid (T and B cell), NK, and myeloid
(macrophage and granulocyte) lineages in a representative mixed
allogeneic chimera prepared using a nonlethal conditioning regimen
(BALB/c.fwdarw.B10). Splenic lymphoid tissue was analyzed 10-12
weeks following reconstitution. Further analysis of recipient and
donor-derived myeloid lineages was performed in the myeloid gate
using biotinylated anti-H-2.sup.b and H-2.sup.d mAb detected using
SA-PE. Macrophages were analyzed using MAC-1 FITC and granulocytes
were detected using GR-1 FITC. The percentage of donor and
recipient-derived cells within each lineage is expressed in the
upper right hand corner of each respective plot. Results are
normalized 100%.
FIG. 7 Survival of full thickness tail skin grafts placed 1 to 7
months post reconstitution using two different donor strain
combinations B10.BR (H-2.sup.k) or BALB/c (H-2.sup.d). Each animal
(n=14) received three skin grafts: recipient-type (B10; H-2.sup.b);
donor-type (B10.BR; H-2.sup.k, or BALB/c; H-2.sup.d ; and third
party (DBA; H-2.sup.d or B10.BR; H-2.sup.k). Survival was
calculated by the life table method. Grafts were followed for a
minimum of 35 days. Grafts were scored for evidence of rejection,
which was considered complete when no viable residual could be
detected.
FIG. 8A-8C Specific CTL lysis of .sup.51 Cr-labelled target in
one-way CML towards recipient (B10), donor (B10.BR), and
third-party (BALB/c) targets. Spontaneous release was <25%
unless otherwise indicated. One of five representative
experiments.
FIG. 9 Percentage of animals with allogeneic donor cell engraftment
after treatment with various cytoablative agents.
FIG. 10 Percentage of donor cell engraftment in mice engrafted with
allogeneic bone marrow cells after treatment with CyP, ALG and
various doses of TBI. B10 mice were transplanted with
15.times.10.sup.6 B10.BR cells.
FIG. 11 Percentage of allogeneic donor cell engraftment in mice
treated with: 1=3Gy TBI, 2=ALG (2 mg)+3Gy TBI, 3=3Gy TBI+CyP (200
mg/kg), 4=ALG (2 mg)+CyP (200 mg/kg), 5=ALG (2 mg)+3Gy TBI+CyP (200
mg/kg)
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to non-lethal methods of conditioning
a recipient for bone marrow transplantation. These methods include
the use of non-lethal doses of irradiation, cell type-specific
antibodies and active fragments thereof, cytotoxic drugs or a
combination thereof. In particular, the present invention
encompasses an approach to make space in a recipient's bone marrow
by targeting critical cell populations in the hematopoietic
microenvironment in the complete absence of radiation
treatment.
The invention is discussed in more detail in the subsections below,
solely for the purpose of description and not by way of limitation.
For clarity of discussion, the specific procedures and methods
described herein are exemplified using animal models; they are
merely illustrative for the practice of the invention. Analogous
procedures and techniques are equally applicable to all mammalian
species, including human subjects.
5.1. NON-LETHAL CONDITIONING REGIMENS FOR DONOR CELL
ENGRAFTMENT
Mixed allogeneic chimerism has been demonstrated to be an effective
means to induce donor-specific transplantation tolerance and
preserve immunocompetence. Unlike fully allogeneic chimeras, which
are relatively immunoincompetent, mixed allogeneic chimeras in
which both host and donor-derived bone marrow cells co-exist,
exhibit superior immunocompetence because of the presence of both
host and donor-derived cells (Singer et al., 1981, J Exp Med 1-3:
1286; Ildstad et al., 1985, J Exp Med 162: 231). Mixed chimerism
has been achieved using two different approaches, (1) high dose
total lymphoid irradiation (TLI) followed by donor bone marrow
transplantation (Slavin et al., 1978, J Exp Med 147(4): 963) or (2)
total body irradiation (TBI) followed by the transplantation of a
mixture of T-cell depleted syngeneic and allogeneic bone marrow
cells (Singer et al., 1981, J Exp Med 1-3: 1286; Ildstad et al.,
1985, J Exp Med 162: 231). Both approaches result in stable
long-term syngeneic and allogeneic chimerism and are associated
with donor specific transplantation tolerance to skin and solid
organ grafts (Ildstad and Sachs, 1984, Nature 307: 168). The
application of mixed allogeneic chimerism to induce tolerance
clinically has been significantly hampered, however, by the
excessive morbidity and cytoreduction which is believed to be a
prerequisite for allogeneic engraftment across multimajor
histocompatibility barriers.
Both host and donor factors are known to influence engraftment.
Stable engraftment requires the host to "tolerate" the allogeneic
stem cell and provide hematopoietic niches for the allogeneic stem
cells to engraft, proliferate, and differentiate. These two
conditions, believed to be essential for the engraftment of the
stem cell, are referred to as (1) immunosuppression and (2)
cytoreduction (Cobbold et al., 1992, Immunol Rev 129: 165).
Radiation-based regimens optimize both of these requirements by
removing radiosensitive components within the recipient bone marrow
to "make space" and by providing a generalized
immunosuppression.
The efficacy and necessity of TBI in the facilitation of bone
marrow engraftment have been demonstrated in a number of syngeneic
and allogeneic models (Down et al., 1991, Blood 77(3): 661). In
earlier studies by Down et al, even syngeneic engraftment failed to
occur in a murine model without some pretreatment of the recipient
with TBI (Down et al., 1991, Blood 77(3): 661). Minimal space and
suppression were required for syngeneic reconstitution since
partial engraftment occurred with as little as 2Gy. However,
significantly greater immunosuppression and/or "hematopoietic
space" was required for MHC identical but minor antigen mismatched
allogeneic marrow, resulting in failure of engraftment with less
than 5.5Gy of TBI (Down et al., 1991, Blood 77(3): 661). The
dose-response curve of engraftment versus radiation dose in these
previous MHC-compatible studies was sigmoidal, with a steep
increase in the percentage of allogeneic engraftment seen at doses
of 6 Gy or greater. The immunologic resistance to MHC-compatible
allogeneic engraftment is nearly identical to the sigmoidal
dose-response curve seen for MHC-disparate bone marrow engraftment
in the present radiation-based conditioning model for both
allogeneic and xenogeneic combinations (FIG. 1). The percentage of
animals which engraft with a given radiation dosage undergoes an
abrupt transition from no alloengraftment to nearly complete
allochimerism within a very precise and reproducible range of 5.5Gy
to 7Gy of TBI. The curve is shifted slightly to the right for
xenoengraftment. These data therefore support the concept that
there is a form of "space-making" provided by irradiation
treatment, since at 5.5Gy only 10% of animals engrafted while at
6Gy.gtoreq.60% engrafted. A difference of 0.5Gy would be unlikely
to represent a differential immunosuppressive effect, since NK
cells and lymphocytes have a low threshold of radiosensitivity.
There is a well-characterized relative resistance to engraftment of
the bone marrow stem cell across allogeneic disparities (Vallera
and Blazer, 1989, Transplantation 47: 70-1). Three times more
allogeneic bone marrow cells are required to achieve reliable
engraftment compared with autologous or syngeneic reconstitution
(Ildstad and Sachs, 1984, Nature 307: 168). Resistance to
engraftment is further increased in donor-recipient strain
combinations in which both MHC and minor antigen disparities exist
and even further for xenoengraftment; i.e. Rat.fwdarw.mouse is
eight times more, and human.fwdarw.mouse is ten times more (Ildstad
et al., 1991, J. Exp. Med. 174: 467). In the present invention,
engraftment of MHC and minor antigen-disparate bone marrow occurred
less often than did engraftment of MHC-disparate but minor antigen
congenic bone marrow in recipients conditioned with a similar dose
of TBI. These data indicate that the radioresistance of the barrier
to alloengraftment increases with increasing antigenic
disparity.
It has been established in Section 6, infra, that alloengraftment
can be maximized yet recipient morbidity and mortality minimized by
the addition of ALG or CyP to radiation-based conditioning. When
5Gy of TBI was administered in combination with either ALG or CyP,
stable engraftment of allogeneic donor bone marrow cells was
achieved. However, immunosuppression alone, without TBI, or TBI
alone at a dose of 5Gy, were not sufficient for alloengraftment.
Furthermore, CyP was equally effective in enhancing allogeneic
engraftment when given before or shortly after bone marrow
transplantation, in conjunction with low dose TBI. When ALG and CyP
are used in combination with TBI, the dosage of TBI necessary to
achieve stable donor cell engraftment is substantially reduced to
2Gy or lower. At 3 Gy, there is significant and stable engraftment
in most recipients conditioned by the combination treatment.
TBI may be administered in a modified manner in the form of TLI.
TLI is delivered in the same fashion as TBI, except that the entire
body of the recipient is not exposed. The irradiation is directed
at lymphoid tissues such as the spleen, vertebral column, sternum,
ribs, etc. As a result, TLI is, in essence, a partial TBI that is
less aggressive and cyto-ablative, and thus higher doses may be
administered without lethal effects. TLI conditioning may be
supplemented by CyP and/or ALG. These agents may be given before or
after TLI. Preferably, they should be administered prior to TLI,
and at one or more doses.
Historically, TLI has been utilized in fractionated doses to treat
cancer patients. Typically, about 20Gy is administered in
approximately 10 divided doses at 2Gy/dose. However, a single and
relatively high (.gtoreq.7.5 Gy) dose of TLI as a conditioning
regimen has not been studied for conditioning recipients. Section
8, infra, shows that a single dose of TLI may lead to low levels of
donor cell engraftment in a small percentage of recipients.
However, the combined use of TLI with an alkylating agent such as
CyP results in up to 30% of donor cell engraftment in baboons,
demonstrating in vivo efficacy in non-human primates. Similarly,
TLI may also be used with an antibody such as ALG or an antibody
that is directed to stromal cells. The combined use of TLI,
antibody and alkylating agent may further reduce the necessary dose
of TLI.
The importance of the hematopoietic niches or "space" contributed
by the low dose of TBI is even more evident when TBI is given one
week prior to bone marrow transplantation, since engraftment did
not occur in that setting. This failure to engraft is probably not
due to loss of the immunosuppressive effect of the radiation, since
suppression of T cell function following a single dose of radiation
has been demonstrated to persist for months or even years (Haas et
al., 1985, Trans Proc 17(1): 1294). Rather, it is highly likely
that the making of space is a prerequisite for engraftment and the
delay between TBI and transplantation allowed the host marrow to
undergo radiation repair, occupy the available spaces created by
the radiation, and prevent alloengraftment despite adequate
immunosuppression by ALG. Repair of sub-lethal damage, resulting in
a similar dose-sparing effect, has been documented with
fractionated TBI in syngeneic and MHC-compatible models (Down et
al., 1991, Blood 77(3): 661). This repair results in a greater
resistance to alloengraftment with a shift in the radiation
dose-response curve requiring an additional 3Gy of initial
radiation to induce donor chimerism (Down et al., 1991, Blood
77(3): 661).
It is of note that the same failure of alloengraftment did not
occur if TBI is given one week prior to allogeneic bone marrow
transplantation and followed by CyP treatment. Unlike ALG, which is
believed to be immunosuppressive but not cytoreductive, CyP is
toxic to rapidly proliferating cells. This toxicity may, therefore,
have prevented the repair of sublethal damage to hematopoietic
niches and syngeneic repopulation necessary to resist
alloengraftment. In addition, CyP has been shown to result in
endothelial injury with subsequent loss in the integrity of the
sinus endothelial barrier (Shirota and Tavassoli, 1991, Exp.
Hematol. 19: 369). The augmentation of donor chimerism seen with
CyP, as compared to ALG, therefore, may be secondary to increased
access to hematopoietic niches rather than to any increase in the
amount of unoccupied space.
The induction of tolerance towards MHC-disparate grafts using mAb
therapy was recently reported (Cobbold et al., 1990, Eur J Immunol
20: 2747). However, tolerance to other tissues of donor organ, i.e.
splenocytes or bone marrow, was not reliably induced without the
addition of 6Gy TBI. Moreover, engraftment was variable and often
transient. This disparity in tolerance for different tissues has
been termed "split tolerance". These recipients exhibit "tolerance"
towards a local form of donor antigen, i.e. skin graft, but often
exhibit proliferative and cytotoxic reactivity to other donor
tissues such as lymphoid cells.
Although split tolerance has been a limitation in several nonlethal
conditioning regimens, the preparation of allogeneic chimeras using
low dose TBI-regimens in the present invention have resulted in
systemic donor-specific tolerance towards both skin grafts and
lymphoid tissues of donor-type. The prolonged survival of
donor-type skin grafts in all animals which exhibit successful
engraftment of allogeneic bone marrow is donor-specific, since
chimeras are immunocompetent to reject third-party skin grafts with
a time course similar to unmanipulated control mice. Similarly,
animals which exhibit any degree of donor chimerism also exhibit
specific functional tolerance in vitro towards donor antigens on
lymphoid tissues as assessed by in vitro assays. No evidence of
split tolerance has been found in any of the allogeneic chimeras
tested, as animals which fail to exhibit tolerance towards donor
lymphoid tissues also reject donor skin grafts and contain no
detectable donor chimerism. In the present invention, chimerism is
always associated with stable functional donor-specific
transplantation tolerance in vivo and in vitro.
The mixed chimeras prepared with the nonlethal approaches
characterized in the studies described herein exhibit similar
multilineage donor chimerism which is stable for the duration of
follow-up (.gtoreq.8 months). Significant levels of donor chimerism
are detected within each of the various lineages including lymphoid
(T and B lymphocytes), NK cell, and myeloid (macrophages,
granulocytes, erythrocytes, and platelets) in almost all animals
examined (n=10). The level of donor chimerism among each of the
lineages is variable within individual animals, an observation
which parallels the findings in mixed chimeras prepared with lethal
conditioning. These data suggest that tight regulatory control over
both syngeneic and allogeneic pluripotent stem cells exists which
determines the level of production of each individual lineage.
Moreover, lineage production is also influenced by the conditioning
used, since non-lethal mixed xenogeneic (Rat.fwdarw.mouse) chimeras
produced rat-derived red blood cells, while chimeras prepared by
lethal conditioning do not. There is also substantial data to
suggest that the hematopoietic microenvironment in which the stem
cells reside, may profoundly influence the development of the stem
cells into various cell lineages.
The specificity of this regulation is clearly evident on
examination of those chimeras which produce erythrocytes of only
donor origin, despite an intact host hematopoietic system and
production of syngeneic cells within the other hematolymphopoietic
lineages. Such regulation may require specific cell-cell
interactions found within "hematopoietic niches", thereby
explaining the necessity of "space-making" agents, such as
radiation, in allogeneic marrow transplantation. Recent studies by
Jacobsen (Jacobsen et al., 1992, J Exp Med 176: 927) have shown the
specific cell-cell interactions within murine bone marrow between B
cell precursors and a stromal cell. Each lineage may have a limited
number of specific stromal cells necessary for developmental
maturation or, alternatively, a single cell may be regulated to
favor differentiation of a certain lineage at a given time. Prior
to the present invention, methods to specifically target the cells
which constitute the hematopoietic niches have not been
attempted.
Nonlethal conditioning approaches which result in multilineage
mixed chimerism may significantly expand the application of bone
marrow transplantation for non-malignant diseases. Hematologic
abnormalities including thalassemia and sickle cell disease,
autoimmune states, and several types of enzyme deficiency states
have previously been excluded from bone marrow transplantation
strategies because the high morbidity and mortality associated with
conditioning to achieve fully allogeneic bone marrow reconstitution
could not be justified (Kodish et al., 1991, N Engl J Med 325(19):
1349). Sickle cell disease is a prime candidate for mixed
allogeneic reconstitution since only 40% of normal erythrocytes are
required to prevent an acute crisis (Jandl et al., 1961, Blood
18(2):133; Cohen et al., 1992, Blood 76(7): 1657).
In the present invention, multilineage mixed chimerism has been
reliably achieved using minimal conditioning of the recipient.
Other models of engraftment using sublethal recipient conditioning
have failed to establish the presence of stable multilineage mixed
allogeneic chimerism and permanent donor-specific tolerance which
is crucial for conditions such as sickle cell disease or
thalassemia. The nonlethal conditioning approaches described
herein, may be useful in the treatment of non-fatal hematologic
abnormalities, as well as for the induction of tolerance to
simultaneous or subsequent cellular or solid organ allografts, in
which the morbidity of conventional full cytoreduction is
prohibitive.
5.2. ANTIBODY FOR USE IN CONDITIONING
The hematopoietic microenvironment is primarily composed of
hematopoietic cells and stromal cells. The stromal cells occupy
much space of the bone marrow environment and they include
endothelial cells that line the sinusoids, fibroblastic cells such
as adventitial reticular cells, perisinusoidal adventitial cells,
periarterial adventitial cells, intersinusoidal reticular cells and
adipocytes, and macrophages (Dorshkind, 1990, Annu. Rev. Immunol.
8: 111; Greenberger, 1991, Crit. Rev. Oncology/Hematology 11: 65).
In addition, the Applicant has recently identified, characterized
and purified a previously unknown cell type from the bone marrow
that facilitates the engraftment of bone marrow stem cells across
allogeneic and xenogeneic barriers. This cell referred to as
hematopoietic facilitatory cell must be matched with the stem cell
at the MHC for it to enhance stem cell engraftment. The
facilitatory cells express a unique profile of cell surface
markers: Thy-1.sup.+, CD3.sup.+, CD8.sup.+, CD45.sup.+ CD45R.sup.+,
MHC class II.sup.+, CD4.sup.-, CD5.sup.-, CD14.sup.-, CD16.sup.-,
CD19.sup.-, CD20.sup.-, CD56.sup.-, .gamma..delta.-TCR.sup.- and
.alpha..beta.B-TCR.sup.-. These cells are a newly recognized
stromal cell population that is a critical component of the
hematopoietic microenvironment. In allogeneic reconstitution
experiments in mice, the murine facilitatory cells have been shown
to be radiosensitive at about 3Gy.
The various stromal cell types express a number of
well-characterized surface markers, including but not limited to,
vascular addressing, mannosyl and galactosyl residues, fasciculin
III, villin, tetrapeptide, neural cell adhesion molecule receptor,
hemonectin, B1 integrins, B2 integrins and B3 integrins
(Greenberger, 1991, Crit. Rev. Oncology/Hematology 11: 65). All the
stromal cell populations including the facilitatory cells are
potential targets of the conditioning regimen for recipients that
is necessary for successful donor cell engraftment. Therefore,
antibodies reactive with or specific for stromal cell surface
markers may be used to deplete stromal elements in a cell
type-specific non-lethal conditioning approach to make space
available for bone marrow transplantation. For example, antibodies
directed to Thy-1, MHC Class I and Class II molecules expressed on
many stromal cell types may be used for this purpose. In addition,
a monoclonal antibody designated STRO-1 has been shown to react
with a cell surface antigen expressed by stromal elements in human
bone marrow (Simmons and Torok-Storb, 1991, Blood 78: 55). This
antibody may be particularly useful for depleting stromal cells for
making space in the bone marrow. In the mouse model, anti-Thy-1 and
rabbit-anti-mouse-brain (RAMB) antibodies are effective in removing
the facilitatory cell population from the bone marrow. RAMB is a
polyclonal serum prepared by immunizing rabbits with homogenized
mouse brain (Auchincloss and Sachs, 1983, Transpl. 36: 436). Human
brain also contains a number of epitopes cross-reactive with those
expressed by the facilitatory cells. Thus, rabbit-anti-human-brain
antibodies have been produced and may be used to remove the
facilitatory cells from the hematopoietic microenvironment.
However, since murine facilitatory cells have been shown to be
radiosensitive at about 3Gy but substantial donor cell engraftment
does not occur at radiation doses less than 6Gy as shown herein in
Section 6, infra, it is possible that the elimination of stromal
cell types other than facilitatory cells is necessary to create the
greatest amount of space for optimal donor cell engraftment.
Also within the scope of the invention is the production of
polyclonal and monoclonal antibodies which recognize novel antigens
expressed by stromal cells including the facilitatory cells of the
hematopoietic microenvironment for use as specific agents to
deplete these cells.
Various procedures known in the art may be used for the production
of polyclonal antibodies to antigens of stromal cells including
facilitatory cells. For the production of antibodies, various host
animals can be immunized by injection with purified or partially
purified stromal cells including but not limited to rabbits,
hamsters, mice, rats, etc. Various adjuvants may be used to
increase the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
A monoclonal antibody to antigens of stromal cells may be prepared
by using any technique which provides for the production of
antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally
described by Kohler and Milstein (1975, Nature 256: 495-497), and
the more recent human B-cell hybridoma technique (Kosbor et al.,
1983, Immunology Today 4: 72; Cote et al., 1983, Proc. Natl. Acad.
Sci., USA 80: 2026-2030) and the EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96). Techniques developed for the production of
"chimeric antibodies" by splicing the genes from a mouse antibody
molecule of appropriate antigen specificity together with genes
from a human antibody molecule can be used (e.g., Morrison et al.,
1984, Proc. Natl. Acad. Sci. USA, 81: 6851-6855; Neuberger et al.,
1984, Nature, 312: 604-608; Takeda et al., 1985, Nature 314:
452-454). Such chimeric antibodies are particularly useful for in
vivo administration into human patients to reduce the development
of host anti-mouse response. In addition, techniques described for
the production of single chain antibodies (U.S. Pat. No. 4,946,778)
can also be adapted.
Antibody fragments which contain the binding site of the molecule
may be generated by known techniques. For example, such fragments
include but are not limited to: the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments (Antibody: A Laboratory
Manual, 1988, Harlow and Lane, Cold Spring Harbor).
5.3. USES OF ANTIBODIES TO STROMAL CELLS
The specific embodiments described in Section 6, infra, demonstrate
that non-lethal conditioning of a recipient may be achieved by a
reduced dose of TBI. Further, similar results can be obtained by an
even lower dose of irradiation when applied in combination with ALG
or an alkylating agent. Thus, it is possible to develop a
non-lethal conditioning method by totally eliminating the use of
radiation or chemotherapeutic agents to and by using antibodies to
deplete the critical targets of TBI. A likely target of such an
approach is the various stromal cell populations that form the
hematopoietic microenvironment. Antibodies directed to cell surface
markers of stromal cells may be used to specifically deplete these
cells without other adverse side effects in preparing a recipient
for bone marrow transplantation in the absence of lethal doses of
irradiation. Alternatively, such antibodies may be used in
conjunction with low doses of irradiation and/or cytotoxic
drugs.
According to this embodiment, the antibodies of the present
invention can be modified by the attachment of an antiproliferative
or toxic agent so that the resulting molecule can be used to kill
cells which express the corresponding antigen (Vitetta and Uhr,
1985, Annu. Rev. Immunol. 3: 197-212). The modified antibodies may
be used in the preparation of a recipient prior to bone marrow
transplantation in order to deplete stromal cells to make space for
donor cells to engraft.
Accordingly, the antiproliferative agents which can be coupled to
the antibodies of the present invention include but are not limited
to agents listed in Table 1, infra, which is derived from Goodman
and Gilman, 1990, The Pharmacological Basis of Therapeutics, Eighth
Edition, Pergamon Press, N.Y., pp. 1205-1207, which is incorporated
by reference herein.
Such antibody conjugates may be administered to a human patient
prior to or simultaneously with donor cell engraftment. It is
preferred that these conjugates are administered intravenously.
Although the effective dosage for each antibody must be titrated
individually, most antibodies may be used in the dose range of 0.1
mg/kg--20 mg/kg body weight. In cases where sub-lethal doses of
irradiation are used, TLI of a human recipient may be administered
at 5 to 10 Gy as a single dose or a combined total of 22Gy
administered in fractionated doses. Preferably, TLI may be used
between 7.5-9.5 Gy. Alternatively, TBI may be administered between
5 Gy and 7 Gy.
TABLE 1 ______________________________________ ANTI-PROLIFERATIVE
AGENTS WHICH CAN BE COUPLED TO ANTIBODIES Class Type Agent
______________________________________ Alkylating Agents Nitrogen
Mustards Mechlorethamine Cyclophosphamide Ifosfamide Melphalan
Chlorambucil Ethylenimine Hexamethyl-melamine Derivatives Thiotepa
Alkyl Sulfonates Busulfan Nitrosoureas Carmustine Lomustine
Semustine Streptozocin Triazenes Dacarbazine Antimetabolites Folic
Acid Analogs Methotrexate Pyrimidine Analogs Fluorouracil
Floxuridine Cytarabine Purine Analogs Mercaptopurine Thioguanine
Pentostatin Natural Products Vinca Alkaloids Vinblastine
Vincristine Epipodophyllotoxins Etoposide Teniposide Antibiotics
Dactinomycin Daunorubicin Doxorubicin Bleomycin Plicamycin
Mitomycin Enzymes L-Asparaginase Miscellaneous Agents Platinum
Cisplatin Coordinated Complexes Carboplatin Anthracenedione
Mitoxantrone Substituted Urea Hydroxyurea Methyl Hydrazine
Procarbazine Derivative Adrenocortical Mitotane Suppressant
Aminoglutethimide Hormones and Adrenocorticosteroids Prednisone
Antagonists Progestins Hydroxyprogesterone caproate
Medroprogesterone acetate Megestrol acetate Estrogens
Diethylstilbestrol Ethinyl estradiol Antiestrogen Tamoxifen
Androgens Testosterone propionate Fluoxymesterone Radioactive
Isotopes Phosphorous Sodium phosphate .sup.32 P Iodine Sodium
idoine .sup.131 I Toxins Ricin A chain Diphtheria toxin Pseudomonas
exotoxin A ______________________________________
Any method known in the art can be used to couple the antibodies to
an antiproliferative agent, including the generation of fusion
proteins by recombinant DNA technology (Williams et al., 1987,
Protein Engineering 1: 493).
6. EXAMPLE: ALLOGENEIC BONE MARROW CELLS ENGRAFT IN RECIPIENTS
CONDITIONED BY NON-LETHAL METHODS
6.1. MATERIALS AND METHODS
6.1.1. ANIMALS
Male C57BL/10SnJ (B10), B10.BR, and BALB/c mice 6-8 weeks old were
purchased from the Jackson Laboratory, Bar Harbor, Me. Animals were
housed in a specific pathogen-free facility at the Biomedical
Science Tower at the University of Pittsburgh.
6.1.2. FLOW CYTOMETRY
Recipients were characterized for donor cell engraftment using flow
cytometry (FACS II, Becton Dickinson; Mountain View, Calif.) to
determine the percentage of peripheral blood lymphocytes bearing
H-2.sup.b, H-2.sup.k, and H-2.sup.d encoded antigens as described
(Jeffries et al., 1985, J Exp Med 117: 127). Briefly, peripheral
blood was collected into heparinized plastic serum vials. 200 .mu.l
of Medium 199 (Gibco Laboratories; Grand Island, N.Y.) were added
to each vial. After thorough mixing, the suspension was layered
over 1.5 ml of room temperature Lymphocyte Separation Medium (LSM)
(Organon Teknika; Durham, N.C.) and centrifuged at 37.degree. C.
(400g.times.20 minutes). The buffy coat layer was aspirated from
the Medium 199-LSM interface and washed with medium. Lymphocytes
were stained for class I antigens with anti-H-2.sup.b -FITC
(Pharmingen; San Diego, Calif.), anti-H-2.sup.k -FITC (Pharmingen),
and anti-H-2.sup.d -FITC (Pharmingen) monoclonal antibodies (Mab)
for 45 minutes at 4.degree. C. Lineage typing was performed by two
color flow cytometry using anti-B-cell (B220-FITC, Pharmingen),
anti-T cell (.alpha..beta.-TCR-PE, CD4-FITC, CD8-PE, Pharmingen),
anti-natural killer cell (NK1.1-PE, Pharmingen), anti-granulocyte
(GR-1-FITC, Pharmingen), and anti-monocyte/macrophage (MAC-1-FITC,
Boehringer Mannheim; Indianapolis, Ind.) Mab. These
lineage-specific Mab were displayed versus anti-host (H-2.sup.b,
Pharmingen) and anti-donor (H-2.sup.d or H2.sup.k, Pharmingen) Mab
conjugated to FITC or were biotinylated and detected with a second
streptavidin antibody conjugated to (phycoerythrin PE)
(Pharmingen). Analyses were performed using forward and side
scatter characteristics for the lymphoid and myeloid gates.
6.1.3. PLATELET ISOLATION
Peripheral blood (0.9ml) was collected into heparinized
microcentrifuge vials. The blood was spun for four seconds at the
maximal setting (14,000 rpm) of an Eppendorf microcentrifuge
(Beckman #5415). This setting was chosen through an optimization
strategy in which force and times were varied as a function of
retrieved platelet number. This duration included the acceleration
phase, which is incomplete when power is curtailed at the four
second mark. After this, the samples were allowed to slow to a halt
without braking. Platelet-rich plasma was then carefully aspirated
with a disposable polyethylene pipette, avoiding any disturbance of
the buffy coat. Triplicate platelet counts were obtained using a
Coulter Model ZB1 counter (Hialeah, Fla.), and the average
(variation 5%) calculated. Platelets were then processed as
described for the glucose phosphate isomerase-1 assay, infra.
6.1.4. GLUCOSE PHOSPHATE ISOMERASE-1 (GPI-1) ASSAY
Typing of red blood cell (RBC) and platelet phenotypes was
performed using the GPI-1 assay (Ildstad, et al., 1991, J Exp Med
174: 467). The precipitation pattern for BALB/c mouse and B10 mouse
were performed as controls and determined to be totally disparate.
Briefly, 8 .mu.l of RBC were lysed in 400 .mu.l of distilled water,
and electrophoresis was performed on a Titan III cellular acetate
strips with tris Hcl, 20 mM glycerin, 200 mM buffer (pH 8.7) (200 V
for 1 hr.). Application was 2 cm from the anode. After the run, the
strips were covered with a 1% agarose gel containing Tris-Hcl 100
Mm (pH 8.0), NADP 300 .mu.M, glucose-6-phosphate dehydrogenase 0.5
U/ml, fructose-6-phosphate 50 Mm, MMT 500 .mu.M and phenozine
methosulphate 200 .mu.M. As precipitation occurred with the
formation of formazan salt, the bands became visible (blue). The
gel was removed, the reaction was arrested by immersing the strips
in 5% acetic acid, and the bands were scanned with a Quick-Scan
scanner. Percentages were determined by comparison with the
positive control. Values for each animal were normalized to 100%.
In titrations performed to determine the sensitivity of this assay,
as low as 2% of BALB/c RBC titrated into normal B10 RBC could be
reliably detected (Ildstad, et al., 1991, J Exp Med 174: 467).
After isolation, platelets were typed in a similar fashion.
6.1.5. SKIN GRAFTING
Skin grafting was performed by a modification of the method of
Billingham and Medawar as previously described (Rappaport, 1977,
Trans Proc 9: 894; Kunst et al., 1989, Immunogenetics 30: 187).
Full thickness skin grafts were harvested from the tails of
C57BL/10SnJ (H-2.sup.b), B10.BR (H-2.sup.k), BALB/C (H-2.sup.d),
and DBA (H-2.sup.d) mice. Mice were anesthetized with 0.1% Nembutal
(Abbott Laboratories; North Chicago, Ill.) intraperitoneally and
full thickness graft beds were prepared surgically in the lateral
thoracic wall. Care was taken to preserve the panniculus carnosum.
The grafts were covered by a double layer of vaseline gauze and a
plaster cast to prevent shearing. Three skin grafts from syngeneic,
allogeneic donor, and third-party animals were placed on each
animal with separation of each defect for graft placement by a 3 mm
skin bridge. Casts were removed on the eighth day. Grafts were
scored daily for percent rejection, and rejection was considered
complete when no residual viable graft could be seen. Chronic
rejection was the time point at which erythema and induration
appeared in the grafts. Graft survivals were calculated by the
life-table method (Gehan, 1969, J Chronic Dis 21: 629) and the
median survival time (MST) was derived from the time point at which
50% of grafts were surviving.
6.1.6. MIXED LYMPHOCYTE REACTIONS (MLR)
Mixed lymphocyte reactions were performed as described (Schwartz et
al., 1976, J Immunol 116: 929; Hoffman et al., 1990, J Immunol 145:
2220). Briefly, murine splenocytes were ACK-lysed (ammonium
chloride potassium carbonate lysing buffer), washed, and
reconstituted in DMEM (Gibco Laboratories) supplemented with 0.75%
normal mouse serum, 0.55 mM L-arginine HCl+13.6 .mu.M folic acid
+0.3 mM L-asparagine+10 mM HEPES buffer, 1 mM sodium pyruvate, 2 mM
glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin, 0.05 mM
2-mercaptoethanol and 1 mM N.sup.G mono-methyl L-arginine (Hoffman
et al., 1990, J Immunol 145: 2220 ). 4.times.10.sup.5 responders
were stimulated with 4.times.10.sup.5 irradiated stimulators (20Gy)
in a total of 200.mu.l of media. Cultures were incubated at
37.degree. C. in 5% CO.sub.2 for 4 days, pulsed on the third day
with 1 .mu.Ci [.sup.3 H]thymidine (New England Nuclear; Boston,
Mass.) and harvested on the fourth day with an automated harvester
(MASH II; Microbiological Associated, Bethesda, Md.).
6.1.7. CELL-MEDIATED LYMPHOLYSIS (CML)
CML assays were performed using a modification of techniques as
described (Schwartz et al., 1976, J Immunol 116: 929; Epstein et
al., 1980, J Immunol 125: 129; Lang et al., 1981, Trans Proc 13:
1444). RPMI 1640 medium (Gibco Laboratories) was supplemented as
above, except that 10% fetal calf serum (Gibco Laboratories) was
used in place of normal mouse serum. 4.times.10.sup.6 responders
were co-cultured with 4.times.10.sup.6 irradiated splenocyte
stimulators (20Gy) in 2 ml of medium at 37.degree. C. for 5 days.
Mouse target blasts were stimulated with concanavalin A (Con A)
(Miles Yeda Research Products, Rehovot, Israel) for 2-3 days. After
5 days responders were harvested, counted, and resuspended at
appropriate effector-to-target ratios with 1.times.10.sup.4 51
Cr-labeled, 2-3-d Con A mouse splenocyte blasts. After 4.5 hours,
supernatants were harvested with the Titertek supernatant
harvesting system and specific lysis was calculated as follows:
specific lysis=(experimental release-spontaneous release)/(maximal
Hcl release-machine background).times.100. Spontaneous release was
<25% of maximum release unless otherwise indicated.
6.2. RESULTS
6.2.1. ALLOGENEIC ENGRAFTMENT WITH NONLETHAL TOTAL BODY IRRADIATION
ALONE: DOSE-TITRATION OF RADIATION-BASED CONDITIONING
In other studies of mixed chimerism, lethal irradiation was
utilized as a conditioning approach and reconstitution consisted of
a mixture of T cell depleted (TCD) syngeneic plus TCD allogeneic
bone marrow cells (Ildstad and Sachs, 1984, Nature 307: 168). In
the present invention, a nonlethal radiation-based approach was
used to achieve stable engraftment of allogeneic hematopoietic stem
cells. In this model, the recipient was not fully cytoablated prior
to allogeneic bone marrow transplantation, allowing the
re-emergence of autologous stem cells within an environment of
newly engrafted allogeneic bone marrow cells. Therefore, mixed
allogeneic chimerism resulted even though only allogeneic bone
marrow was infused as donor.
Titrations were performed to determine the minimum dose of TBI
required to permit reliable engraftment of complete MHC-mismatched
but minor antigen matched allogeneic bone marrow
(B10.BR.fwdarw.B10). The dose of TBI administered directly
correlated with the ability of allogeneic bone marrow cells to
engraft (FIG. 1). Although allogeneic engraftment did not occur in
all animals at doses of TBI below 6Gy, a significant increase in
the number of animals which engrafted as allogeneic chimeras
occurred at 6Gy. At this dose 50% of recipient animals that
received 15.times.10.sup.6 allogeneic bone marrow cells exhibited
donor chimerism (FIG. 1). Allogeneic engraftment was reliably
achieved in 100% of all animals conditioned with 7Gy. It is of note
that most of the animals which engrafted exhibited a high level of
allogeneic donor chimerism.gtoreq.95% (Table 2). Evidently, in this
model, allogeneic stem cells either engraft and result in nearly
total allogeneic chimerism or they completely fail to engraft. The
abrupt transition between failure of allogeneic engraftment to
nearly complete allochimerism occurred near 6Gy, indicating that
the "barrier(s)" to allogeneic chimerism is very specific, but once
overcome, allogeneic engraftment occurs unimpeded.
TABLE 2 ______________________________________ LEVEL OF DONOR
CHIMERISM IN ANIMALS WITH ALLOGENEIC ENGRAFTMENT.sup.a % Donor
Reconstitution TBI Dose Chimera # Chimerism
______________________________________ 15 .times. 10.sup.6
B10.Br.fwdarw.B10 5.5Gy 1 99 6Gy 1 99 2 99 3 68 4 98 5 98 6 97 7Gy
1 97 2 99 3 99 4 100 ______________________________________ .sup.a
PBL typing was performed by flow cytometry 2 months
postreconstitution (B10.BR.fwdarw.B10) using antiH2.sup.kFITC
(B10.BR) an antiH-2.sup.bFITC (B10) mAb. Animals are taken from
those represented in FIG. 1. The percent donor chimerism (% B10.BR)
is shown only for those animals which engrafted at each of the
representative TBI doses. Results are pooled from 2 representative
experiments out of a total of 5, and are normalized to 100%.
Similar studies were performed to examine whether engraftment of
bone marrow from a donor strain (BALB/c; H-2.sup.d) which was
mismatched for MHC plus multiminor histocompatibility antigens
could reliably occur at similar non-lethal doses of TBI. Resistance
to alloengraftment was greater for BALB/c bone marrow than for
MHC-disparate B10.BR bone marrow. Although comparable levels of
engraftment with BALB/c and B10.BR allogeneic marrow occurred after
lethal (9.5Gy) conditioning, less than 20% of recipients pretreated
with 6Gy TBI prior to transplantation with BALB/c bone marrow cells
[BALB/c.fwdarw.B10]exhibited any degree of allogeneic
chimerism.
6.2.2. ENGRAFTMENT OF ALLOGENEIC BONE MARROW IS ENHANCED BY
ANTI-LYMPHOCYTE GLOBULIN
Anti-lymphocyte globulin (ALG) is a polyclonal serum directed to
multiple antigens expressed on lymphocytes which has often been
used as an immunosuppressive agent (Monaco, 1991, Trans Proc 23(4):
2061). It produces a transient ablation of lymphocytes from blood
and tissue. Early studies documented the induction of
donor-specific tolerance in thymectomized mice given ALG plus donor
bone marrow cells, leading to extensive study of its uses in
transplantation (Wood et al., 1971, Trans Proc 3(1): 676). Donor
cell engraftment in these studies was transient, if present at all.
Although further attempts at generating permanent tolerance against
fully allogeneic donor antigens with ALG alone have been less
successful, survival of allografts has been prolonged in several
species using ALG in combination with donor bone marrow cells or
other immunosuppressive agents (Wood et al., 1971, Trans Proc 3(1):
676; Monaco, 1991, Trans Proc 23(4):2061). Therefore, it is
possible that this serum preparation was able to deplete cells,
although inefficiently, in the hematopoietic microenvironment to
create space in a recipient.
To examine whether ALG would enhance the engraftment of allogeneic
bone marrow in the established radiation-based model, recipient B10
mice received one of three conditioning approaches prior to
transplantation with 40.times.10.sup.6 or 15.times.10.sup.6 BALB/c
bone marrow cells: 70 mg/kg i.v. ALG given three days prior to bone
marrow transplantation (Group 1); 5Gy of TBI on the day of
transplantation (Group 2); or both ALG and TBI as administered in
groups 1 and 2 (Group 3). The timing of ALG was chosen to assure
maximum immunosuppression at the time of allogeneic bone marrow
infusion (Wood et al., 1971, Trans Proc 3(1): 676). As in previous
analysis, recipients were peripheral blood leukocyte (PBL)-typed
for evidence of allogeneic engraftment 2 months following bone
marrow transplantation. Allogeneic chimerism occurred in 85% of
recipients conditioned with ALG and TBI (Group 3), while no
evidence of alloengraftment was seen in animals receiving either
ALG or TBI alone (Groups 1 and 2) (FIG. 2).
6.2.3. INFLUENCE OF CELL DOSE IN THE ALLOGENEIC INOCULUM ON
ENGRAFTMENT WITH ALG AND TBI CONDITIONING
It has been demonstrated that a greater number of allogeneic donor
cells are required to achieve reliable engraftment when compared
with syngeneic reconstitution (Ildstad and Sachs, 1984, Nature 307:
168; Ildstad et al., 1986, J Exp Med 163: 1343). This has been
termed alloresistance to engraftment. To examine the influence of
donor cell number on the ability of ALG and TBI to enhance
alloengraftment, dose-titration studies were performed in which the
above established radiation plus ALG conditioning were utilized.
Recipients were conditioned as above prior to receiving
40.times.10.sup.6, 15.times.10.sup.6, or 5.times.10.sup.6 BALB/c
bone marrow cells. The percentage of allogeneic donor-derived cells
detected in the peripheral blood of the recipient (i.e. donor
chimerism) increased in relation to the initial number of donor
cells transplanted (Table 3). All animals appeared healthy and had
no stigmata of GVHD although they had received untreated bone
marrow cells.
TABLE 3 ______________________________________ INFLUENCE OF CELL
DOSE OF ALLOGENEIC BONE MARROW INOCULUM ON THE LEVEL OF DONOR
CHIMERISM.sup.a GROUP RECONSTITUTION ANIMAL % BALB/c PBL
______________________________________ 1 40 .times. 10.sup.6
BALB/c.fwdarw.B10 1 87 2 86 3 87 15 .times. 10.sup.6
BALB/c.fwdarw.B10 1 30 2 71 3 75 4 0 5 .times. 10.sup.6
BALB/c.fwdarw.B10 1 1 2 0 ______________________________________
.sup.a PBL typing was performed by flow cytometry 2 months
postreconstitution using antiH-2.sup.dFITC and antiH-2.sup.bFITC
mAb. Results are from one of three representative experiments and
are normalized to 100%.
6.2.4. ALLOGENEIC ENGRAFTMENT IS ENHANCED BY THE ADDITION OF
CYCLOPHOSPHAMIDE TO THE ESTABLISHED RADIATION-BASED
CONDITIONING
CyP is an alkylating agent used widely in treatment of
lymphohematopoietic malignancies, such as leukemia (Gershwin et
al., 1974, Annals Int Med 80: 531; Copelan and Deeg, 1992, Blood
80(7): 1648). It has been demonstrated to increase leukemic cell
killing and reduce tumor relapse (Copelan and Deeg, 1992, Blood
80(7): 1648). CyP also exhibits immunosuppressive effects, by
killing rapidly proliferating cells and resting lymphoid cells,
with an impairment of both humoral and cellular responses (Mayumi
et al., 1987, Transplantation 44(2): 286). Although conditioning
with CyP alone does not result in allogeneic engraftment,
combination therapies have proven useful in permitting engraftment
of bone marrow from HLA-identical siblings (Graw et al., 1972,
Transplantation 14: 79).
In order to assess the ability of CyP to enhance alloengraftment in
the established radiation-based model, B10 mice were treated with
one of three conditioning approaches prior to transplantation with
40.times.10.sup.6 B10.BR or BALB/c bone marrow cells. Mice received
200 mg/kg i.p. of CyP alone (Group 1); 5Gy of TBI on the day of
transplantation (Group 2); or 5 Gy TBI followed by CyP 2 days later
(Group 3). Animals were PBL typed 2 months following
reconstitution. Engraftment of allogeneic bone marrow occurred in
nearly all recipients receiving 5Gy TBI plus CyP (FIG. 3). The
degree of donor chimerism achieved was >90% in all chimeras
conditioned with this approach. In contrast, all animals treated
with TBI or CyP alone failed to engraft (Groups 1 and 2).
6.2.5. INFLUENCE OF TIMING OF TBI ON ALLOENGRAFTMENT IN RECIPIENTS
CONDITIONED WITH ANTI-LYMPHOCYTE GLOBULIN OR CYCLOPHOSPHAMIDE
To examine the influence of timing of radiation on the engraftment
of allogeneic bone marrow, recipient B10 mice were irradiated with
5Gy TBI one week prior to transplantation with 40.times.10.sup.6
BALB/c allogeneic bone marrow cells. Additional animals, prepared
in an identical fashion, received 70 mg/kg i.v. of ALG three days
prior to transplantation or received 50 mg/kg i.p. CyP six, five,
four, and three days prior to transplantation.
Animals conditioned with 5Gy of radiation alone failed to engraft
even if the radiation was administered one week prior to
transplantation (FIG. 4). Although 75% of the recipients exhibited
allogeneic chimerism when treated with ALG plus TBI administered on
the day of bone marrow transplantation, this enhancement of
alloengraftment did not occur when TBI was given one week prior to
transplantation. In contrast, the timing of TBI had little effect
on the enhancement of alloengraftment seen with CyP. Nearly 75% of
all recipient mice treated with TBI and CyP engrafted regardless of
donor-strain or whether the CyP was administered before or shortly
after the TBI (n=15) (FIG. 4). All of these chimeras exhibited
.gtoreq.90% allogeneic donor chimerism.
All of the above approaches indicate that the hematopoietic
microenvironment plays a major role in bone marrow engraftment.
6.2.6. CHARACTERIZATION OF A NONLETHAL RADIATION-BASED APPROACH FOR
CYTOREDUCTION
To assure that the conditioning described herein was "nonlethal"
with respect to overall morbidity and hematopoietic viability,
control mice were conditioned but did not receive an allogeneic
bone marrow transplant. Survival of the animals was excellent (FIG.
5), and none of the regimens used in this study resulted in any
observable morbidity, i.e. diarrhea, cachexia, lassitude, hunched
gate, dermatitis, alopecia, or anorexia. Moreover, these
conditioning regimens were not lethal to the host hematopoietic
stem cell since autologous repopulation resulted.
6.2.7. NONLETHAL MIXED CHIMERAS: EVIDENCE FOR MULTILINEAGE MIXED
CHIMERISM
Mixed allogeneic chimeras conditioned with lethal TBI (9.5Gy)
exhibit stable mixed chimerism of lymphoid and myeloid lineages,
including T cells, B cells, NK cells, erythrocytes, platelets, and
macrophages. To determine whether mixed allogeneic chimeras
prepared with nonlethal conditioning exhibited selective syngeneic,
allogeneic or mixed chimerism of individual hematolymphopoietic
lineages, studies were undertaken to determine the proportion of
cells within each lineage which were host (B10) or donor
(BALB/c)-derived.
Animals which exhibited evidence for engraftment by PBL typing also
had allogeneic cells of donor origin detected for each of the
individual hematolymphopoietic lineages produced by the stem cell
(FIG. 6A, 6B and 6C). The contribution of donor-derived cells
varied among each of the lineages in the ten animals tested, with T
lymphocytes ranging from 3.6 to 100%; B lymphocytes from 3.8 to
99%; NK cells from 9.8 to 96%; and macrophages from 21 to 76%. It
was also influenced by the conditioning approach utilized.
6.2.8. EVIDENCE THAT ERYTHROCYTES AND PLATELETS IN ALLOGENEIC
CHIMERAS ARE OF BOTH SYNGENEIC AND ALLOGENEIC ORIGIN
In order to analyze the proportion of donor and host erythrocytes
(RBC) and platelets, allogeneic chimeras were prepared using BALB/c
(H-2.sup.d) and B10 (H-2.sup.b) donor/recipient strain combinations
which differ at the Glucose Phosphate Isomerase-1 (GPI-1)
isoenzyme. All except one of the chimeras with known allogeneic PBL
chimerism also exhibited RBC and platelets of allogeneic origin
(Table 4). The proportion of allogeneic chimerism differed between
each of the various lineages in individual animals, suggesting that
the degree of allogeneic chimerism may be independently regulated
for each hematopoietic lineage.
TABLE 4 ______________________________________ PHENOTYPE OF
PLATELETS AND ERYTHROCYTES IN MIXED ALLOGENEIC CHIMERAS.sup.a %
BALB/c TBI-based % BALB/c % BALB/c lymphoid Reconstitution regimen
platelets RBC cells ______________________________________
BALB/c.fwdarw.B10 5Gy + ALG 55 64 86 0 0 30 14 30 71 5Gy + CyP 71
100 99 78 100 98 69 100 91 31 100 92 Normal B10 -- 0 0 0 Normal --
100 100 99 BALB/c ______________________________________ .sup.a One
representative experiment for phenotyping of platelets and
erythrocytes by GPIisomerase assay, and enzyme for which B10 and
BALB/c mice differ. Lymphoid typing was performed by flow cytometry
using antiClass I H2.sup.b and H2.sup.d Mab. Analyses were
performed using the forward and side scatter characteristic for the
lymphoid gate. Results were normalized to 100% Animals were typed 2
months post reconstitution.
The single animal which exhibited lymphoid chimerism without
evidence of allogeneic platelets or erythrocytes demonstrated
stable lymphoid chimerism for .gtoreq.75 days post reconstitution.
The lack of multilineage chimerism may be secondary to selective
lineage regulation or may indicate engraftment of a lymphoid
progenitor rather than engraftment of the pluripotent stem cell
itself. All recipients which failed to exhibit PBL chimerism also
had no evidence for allogeneic chimerism of erythroid or platelet
lineages.
6.2.9. EVIDENCE FOR SPECIFIC TOLERANCE IN VIVO TO DONOR-TYPE SKIN
GRAFTS
Mixed allogeneic chimeras prepared with nonlethal conditioning were
tested for evidence of donor-specific tolerance in vivo by
skin-grafting. B10 recipient mice received full thickness tail skin
grafts of recipient, donor (B10.BR or BALB/c), or third-party
origin (BALB/c, DBA, or B10.BR) 1 to 7 months following nonlethal
conditioning and reconstitution (BALB/c.fwdarw.B10;
B10.BR.fwdarw.B10). Grafts were read blindly and assessed on a
daily basis for signs of rejection. In all recipients there was an
absolute correlation between engraftment and tolerance, since mice
with documented chimerism accepted donor-type skin grafts yet
rejected MHC-disparate third-party skin grafts with a time course
similar to identically-conditioned but unreconstituted controls
(FIG. 7). All recipients which failed to exhibit allogeneic
chimerism (<0.5%) promptly rejected both donor and third-party
skin grafts.
6.2.10. FUNCTIONAL DONOR-SPECIFIC TOLERANCE IN VITRO
Nonlethally conditioned chimeras were assessed for donor-specific
tolerance and immunocompetence in vitro using MLR and CML assays
directed against donor and third-party antigens. Lymphocytes from
chimeras which had evidence for allogeneic engraftment were
functionally tolerant to both host (B10), and donor-strain (B10.BR
or BALB/c) alloantigens but were reactive to third-party
alloantigens in an MLR assay (BALB/c or BR10.BR, respectively)
(Table 5). All similarly treated recipients without detectable
allogeneic chimerism were reactive to both donor and third-party
alloantigens.
Similarly, lymphocytes from recipient animals with allogeneic
chimerism failed to lyse targets with host (B10) or donor (B10.BR)
alloantigens, but were fully capable of third-party (BALB/c) target
lysis in CML (FIG. 8A-8C). Lymphocytes from control animals without
chimerism exhibited reactivity directed against all MHC-disparate
targets.
TABLE 5
__________________________________________________________________________
REACTIVITY OF NONLETHALLY CONDITIONED MIXED ALLOGENEIC CHIMERAS IN
ONE-WAY MLR.sup.a
__________________________________________________________________________
[.sup.3 H]-Thymidine Incorporation (cpm .+-. SEM) Animal Anti-B10
Anti-BR Anti-BALB/c Self anti-self
__________________________________________________________________________
Normal B10 3057 .+-. 133 43,223 .+-. 3,838 58,135 .+-. 3,887 --
Normal B10.BR 40,900 .+-. 241 3,608 .+-. 446 59,537 .+-. 2,510 --
Chimera 1 7,173 .+-. 883 3,5O7 .+-. 208 86,892 .+-. 3,763 2,001
.+-. 127 Chimera 2 5,264 .+-. 886 4,077 .+-. 527 67,019 .+-. 777
3,175 .+-. 105
__________________________________________________________________________
Stimulation Index.sup.b Animal B10 B10.BR BABL/c
__________________________________________________________________________
Normal B10 1.0 14.1 19.0 Normal B10.BR 11.3 1.0 16.5 Chimera 1 3.6
1.7 43.4 Chimera 2 1.7 1.3 21.1
__________________________________________________________________________
.sup.a Mean .+-. SEM of triplicate cultures in 1:1
responderto-stimulator ratio. Animals were tested 2-6 months
following reconstitution. This is one of five representative
experiments. B10.BR bone marrow was infused into B10 recipients for
each of the chimeras shown. .sup.b Stimulation index is a ratio of
the cpm generated in response to a given stimulator over the
baseline cpm generated in response to the host. (Chimera
antistimulator/Chimera antiself)
6.2.11. NONLETHAL PREPARATIVE REGIMENS RESULT IN STABLE ALLOGENEIC
CHIMERISM AND EXCELLENT LONG-TERM RECIPIENT SURVIVAL AND NO
EVIDENCE FOR GVHD
All allogeneic chimeras which engrafted with allogeneic donor bone
marrow (n=51) exhibited excellent survival and early evidence of
donor chimerism by 3.5 to 4 weeks following bone marrow
transplantation. Chimerism remained stable throughout a minimum
follow-up of 3 to 4 months post reconstitution. None of the animals
had evidence of GVHD for up to 8 months in follow-up. The overall
mortality was less than 1%.
6.2.12. ALLOGENEIC ENGRAFTMENT AFTER CONDITIONING WITH NONLETHAL
TOTAL BODY IRRADIATION, ANTI-LYMPHOCYTE GLOBULIN AND
CYCLOPHOSPHAMIDE
The following study was carried out to examine whether the
conditioning of a recipient with the combined treatment of ALG and
CyP would reduce the dosage of TBI necessary to result in stable
engraftment of allogeneic donor cells. B10 mice were treated with
ALG at 2 mg/mouse i.v. at day -3 before bone marrow
transplantation. Then on day 0, the same animals were treated with
various doses of TBI and 15.times.10.sup.6 B10.BR or BALB/c bone
marrow cells, followed by CyP (200 mg/kg) injection two days later.
Mixed allogeneic chimerism was achieved in .gtoreq.90% of the
animals conditioned with 3Gy TBI, ALG and CyP. At 2 Gy TBI, a lower
but significant percentage of recipients were also engrafted with
donor cells (FIG. 9). FIG. 10 shows that even at 2 Gy, the combined
treatment of these regimens allowed a definite percentage of donor
cell engraftment. This TBI dosage could even be reduced to 1Gy if
higher numbers of donor cells were transferred. As the dosage of
TBI increased, there was also a proportional increase of the
percentage of donor cell engraftment in the recipients. FIG. 11
illustrates that when the conditioning was performed at 3Gy of TBI,
the combined use of TBI, ALG and CyP was the only method capable of
producing a substantial percentage of donor cell engraftment.
Although 200 mg/kg of CyP was used as the dose of choice, it was
shown that the entire range of 50-200 mg/kg of CyP was able to
condition a recipient in combination with TBI and ALG. Similarly,
ALG yielded positive conditioning results when administered at
0.5-2 mg/animal. Additionally, a higher number of donor cells
always produced higher levels of engraftment. This was demonstrated
when BALB/c donor cells were used in place of B10.BR. Since BALB/c
cells were incompatible with B10 recipients at both the MHC and
minor antigens, it generally required a stronger conditioning
treatment to achieve BALB/c cell engraftment than that necessary
for B10.BR. This could be accomplished by increasing the dosage of
any one of the three regimens, or alternatively, by a higher number
of donor cells.
The engraftment of donor cells was stable and in diverse blood cell
lineages, including T cells, B cells, NK cells, RBC, granulocytes,
platelets and macrophages. When the animals were transplanted with
skin grafts from the donor, donor-specific transplantation
tolerance was observed, but third party grafts were rejected.
Similar pattern of reactivity was confirmed in MLR and CML. The
combined use of three regimens was non-lethal, since all treated
animals survived for more than 100 days, while all mice treated
with TBI at 9.5Gy died by day 10.
7. EXAMPLE: XENOGENEIC BONE MARROW CELLS ENGRAFT IN RECIPIENTS
CONDITIONED BY NON-LETHAL METHODS
7.1. RESULTS
A similar non-lethal radiation-based model has been established in
which rat bone marrow stem cells engrafted stably (.gtoreq.8
months) in mouse recipients. A sigmoidal curve was also observed
when the percentage of animals with donor cell engraftment was
compared with varying doses of irradiation (FIG. 1). This curve was
shifted slightly to greater radiation doses as compared to the
conditions sufficient for allogeneic engraftment, since only 28.6%
of the animals engrafted at 6.5 Gy. A higher proportion of rat
donor cell engraftment occurred with increasing sub-lethal doses of
radiation. At 7.5 Gy, all mice demonstrated evidence of rat stem
cell engraftment. Again, the animals exhibited multilineage
chimerism, including the presence of rat .alpha..beta.-TCR.sup.+ T
cells, B cells, NK cells, monocytes, platelets and red blood
cells.
In addition, the xenogeneic chimeras also displayed functional
donor-specific tolerance to both host and donor cells, while their
responses to MHC-disparate third-party rat or mouse stimulator
cells remained intact. In vivo, the chimeras accepted xenogeneic
pancreatic islet grafts from the same donors, whereas they readily
rejected third-party rat islets. Thus, the data obtained from
xenogeneic bone marrow transplantation studies confirmed the
successful use of a non-lethal conditioning regimen, indicating the
importance of the hematopoietic microenvrionment in xenogeneic
donor cell engraftment.
8. EXAMPLE: ALLOGENEIC AND XENOGENEIC ENGRAFTMENT AFTER
CONDITIONING WITH TOTAL LYMPHOID IRRADIATION
In addition to TBI, TLI was also tested in conditioning recipients
for bone marrow transplantation. As a single dose, TLI was simply a
modified form of TBI in that the method of delivery was the same
way, except that only certain parts of the recipient's body was
exposed to the irradiation. Since TLI was a less aggressive and
ablative approach, its dosage could be increased up to 10 Gy
without lethal consequences. In the following study, baboons were
treated with a single dose of 7.5 Gy of TLI at day 0 followed by
transfer of allogeneic baboon bone marrow cells with at least one
MHC disparity. In addition, certain animals were further treated
with a single dose of CyP (50 mg/kg) at day +2, or two doses of CyP
at day -3 and -2. The results demonstrate that the majority of
baboons conditioned with 7.5Gy TLI and two doses of CyP produced
stable (.gtoreq.36 weeks) engraftment of up 30% donor cells. TLI
with a single dose of CyP produced stable donor cell engraftment in
about 50% of the treated animals. Several of the engrafted animals
exhibited donor-specific tolerance in MLR assays after three
months. TLI alone gave rise to donor cell engraftment in about 25%
of the recipients. However, the engraftment occurred at very low
levels, which was detectable only by molecular typing
techniques.
Xenogeneic transplantation with human cells was also performed in
baboons conditioned with TLI. Since xenogeneic barriers were
usually more difficult to overcome, a baboon was treated with CyP
at day -3, -2 and -1, and 9.5 Gy TLI on day 0, followed by
22.times.10.sup.8 /kg human vertebral body bone marrow cells that
had been antibody-depleted to remove GVHD-producing cells such as T
cells, B cells and NK cells. The animal produced chimerism with 15%
human cells two months after transplantation, with no GVHD or
significant morbidity.
The present invention is not to be limited in scope by the
exemplified embodiments, which are intended as illustrations of
individual aspects of the invention. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
All publications cited herein are incorporated by reference in
their entirety.
* * * * *